The present invention relates to an improved process for obtaining a precursor of insulin or an insulin derivative having correctly bonded cystine bridges in the presence of cysteine or cysteine hydrochloride and a chaotropic auxiliary.
Human insulin is a protein with two amino acid chains together having 51 amino acid residues. Six cysteine residues are found in the two amino acid chains, each two cysteine residues being bonded to one another via a disulfide bridge. In biologically active human insulin, the A and B chains are bonded to one another via two cystine bridges, and a further cystine bridge is found in the A chain. Within a human insulin molecule, looked at statistically, there are 15 possibilities for the formation of disulfide bridges. In biologically active human insulin, only one of the 15 possibilities is found. The following cysteine residues are linked to one another in human insulin:
A 6-A 11 PA1 A 7-B 7 PA1 A 20-B 19 PA1 Fermentation of the microorganisms--cell disruption--isolation of the fusion protein--cyanogen halide cleavage of the fusion protein--isolation of the cleavage product having the proinsulin sequence--protection of the cystine residues of proinsulin by S-sulfonate groups--chromatographic purification of the S-sulfonate--formation of the correctly bonded cystine bridges--desalting of the proinsulin--chromatographic purification of the proinsulin having correctly bonded cystine bridges--concentration of the proinsulin solution--chromatographic purification of the concentrated proinsulin solution--enzymatic cleavage of the proinsulin to obtain human insulin--chromatographic purification of the resulting human insulin. PA1 (a) mixing an aqueous suspension of the precursor of insulin or an insulin derivative with an amount of cysteine or cysteine hydrochloride which results in approximately 1 to 15 SH residues of the cysteine or cysteine hydrochloride per cysteine residue of the precursor, PA1 (b) introducing the cysteine- or cysteine hydrochloride-containing suspension of the precursor into an approximately 4 to 9 molar solution of the chaotropic auxiliary at a pH of approximately 8 to 11.5 and a temperature of approximately 15.degree. C. to 55.degree. C., keeping the mixture obtained at this temperature for approximately 10 to 60 minutes; and PA1 (c) introducing the mixture at a pH of approximately 8 to 11.5 and a temperature of approximately 5.degree. C. to 30.degree. C. into an amount of water which results in a dilution of the concentration of the cysteine or of the cysteine hydrochloride in the mixture to approximately 1 to 5 mM and of the chaotropic auxiliary to approximately 0.2 to 1.0 M. PA1 a) an amino acid residue from the group consisting of His, Arg and Lys, PA1 b) a peptide having 2 or 3 amino acid residues, comprising the amino acid residue Arg or Lys at the carboxyl end of the peptide, PA1 c) a peptide having 2-35 genetically encodable amino acids, comprising 1 to 5 histidine residues, or PA1 d) OH; PA1 a) a hydrogen atom, PA1 b) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), or PA1 c) a peptide having 2 to 45 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the carboxyl end of the peptide; PA1 a genetically encodable amino acid residue; PA1 a) an amino acid residue from the group consisting of lysine (Lys) and arginine (Arg), PA1 b) a peptide having 2 to 35 amino acid residues, comprising the amino acid residue lysine (Lys) or arginine (Arg) at the N-terminal end and at the carboxyl end of the peptide, or PA1 c) a peptide having 2 to 35 genetically encodable amino acids, comprising 1 to 5 histidine residues; PA1 a) a hydrogen atom, or PA1 b) a peptide having 2 to 25 amino acid residues, comprising the amino acid residue arginine (Arg) at the carboxyl end of the peptide; PA1 a) a hydrogen atom, or PA1 b) a peptide having 2 to 15 amino acid residues, at whose carboxyl end is found an arginine residue (Arg);
The letters A and B represent the respective insulin amino acid chain and the numbers represent the position of the amino acid residue, which is counted from the amino to the carboxyl end of the respective amino acid chain. Disulfide bridges can also be formed between two human insulin molecules such that an incalculable number of different disulfide bridges can easily result.
A known process for the preparation of human insulin is based on the use of human proinsulin. Human proinsulin is a protein having a linear amino acid chain of 86 amino acid residues, the A and B chains of the human insulin being bonded to one another via a C peptide having 35 amino acid residues. The formation of the disulfide bridges found in human insulin takes place via an intermediate, the cysteine residues of the human insulin being provided with a sulfur protective group, e.g. an S-sulfonate (--S--SO3--) group (EP 0 037 255). A process for obtaining proinsulin having correctly bonded cystine bridges is additionally known (Biochemistry, 60, (1968), pages 622 to 629), which starts from proinsulin obtained from porcine pancreas, in which the cysteine residues are present as thiol residues (--SH). The term "correctly bonded cystine bridges" is understood as meaning the disulfide bridges which are found in biologically active insulin from mammals.
Recombinant DNA processes allow precursors of insulin or insulin derivatives, in particular human proinsulin or proinsulin which has an amino acid sequence and/or amino acid chain length differing from human insulin, to be prepared in microorganisms. The proinsulins prepared from genetically modified Escherichia coli cells do not have any correctly bonded cystine bridges. A process for obtaining human insulin using E. coli (EP 0 055 945) is based on the following process steps:
Disadvantages of this process are the number of process steps and the losses in the purification steps, which lead to a low yield of insulin. From the stage of the isolated fusion protein via cyanogen halide cleavage, sulfitolysis and purification of the proinsulin, a loss of up to 40% of the proinsulin product has to be expected (EP 0 055 945). Similarly, high losses can occur in the course of the subsequent required to obtain the final product.
Yield increases in the preparation of human insulin or insulin derivatives by recombinant DNA means can be achieved if the number of process steps necessary can be significantly reduced.
EP 0 600 372 A1 (or U.S. Pat. No. 5,473,049) and EP 0 668 292 A2 disclose an improved process for obtaining insulin or insulin derivatives, in which the insulin precursor or precursor of the insulin derivative whose cystine bridges are not present in correctly linked form is reacted in the presence of a mercaptan, for example cysteine, and of at least one chaotropic auxiliary, for example urea or guanidine hydrochloride, to give an insulin precursor or precursor of the insulin derivative having correctly bonded cystine bridges. In the known process, these proteins are first dissolved in a very low concentration in aqueous solutions of a chaotropic auxiliary or of mixtures of various chaotropic auxiliaries. The protein mixture is then mixed with an aqueous mercaptan solution.
Surprisingly, it has now been found that the yields of correctly folded precursors of insulins or insulin derivatives can be increased and the reaction times for the folding process can be reduced by delaying the dissolution of the precursor in a first step by means of the chaotropic auxiliary. Instead, a mercaptan, such as cysteine or cysteine hydrochloride,is introduced into the aqueous suspension of the precursor in the first step, followed by dissolution of the precursor in a subsequent step by introducing a chaotropic auxiliary, and finally bringing about the correct folding of the precursor by dilution of the mixture to a preferred cysteine or cysteine hydrochloride concentration with an appropriate amount of water.